基于NF-κB与NRF2/ARE通路探索紫杉醇-比伐卢定介入涂层抗血管再狭窄的有效性及分子机制

Efficacy and molecular mechanism of paclitaxel-bivalirudin interventional coating complex against vascular restenosis based on NF-κB and NRF2/ARE pathways

目的明确紫杉醇-比伐卢定介入涂层(Luo Fengning,LFN)对管腔再狭窄的影响及分子机制。方法体内动物实验分组:WT假手术组、WT血管拉伤组、NRF2-/-血管拉伤组、NF-κB-/-血管拉伤组、WT血管拉伤+LFN干预组、NRF2-/-血管拉伤+LFN干预组、NF-κB-/-血管拉伤+LFN干预组;体外细胞实验分组:第一批分组:Control组、LPS造模组、LPS+LFN组、LPS+LFN+NF-κB敲减组、LPS+LFN+IκB敲减组、LPS+LFN+NF-κB过表达组、LPS+LFN+IκB过表达组。第二批分组:Control组、LPS造模组、LPS+LFN组、LPS+LFN+NRF2敲减组、LPS+LFN+Keap1敲减组、LPS+LFN+NRF2过表达组、LPS+LFN+Keap1过表达组。采用HE染色法检测血管组织病理变化、免疫荧光染色检测血管组织增殖活性、CCK-8法检测细胞增殖率、Transwell法检测细胞迁移和侵袭能力、Q-PCR和Western blot法测定血管组织和细胞中NF-κB与NRF2/ARE通路关键靶标及配体的基因和蛋白表达。结果LFN对血管拉伤模型大鼠的血管内膜增生过程具有显著抑制作用,可在有效阻断管腔再狭窄的同时拮抗血栓形成。与WT血管拉伤组相比,LFN干预后可上调IκB、NRF2、NQO-1和HO-1基因与蛋白表达(P<0.05,P<0.01),下调Keap-1基因和蛋白表达量(P<0.05,P<0.01),此调控作用在NRF2-/-突变型大鼠中可被逆转。NF-κB、NRF2及其配体敲减或过表达可影响LFN对HCASMC模型迁移和侵袭能力的拮抗作用,并可不同程度削弱其对细胞内NF-κB与NRF2/ARE通路关键蛋白和基因表达的调控能力。结论LFN抗血管再狭窄具备体内应用有效性,该效应的部分机制是LFN通过对NF-κB和NRF2/ARE通路上核转录因子及其关键配体的表达进行双通道正反两个方向的统一调控而实现。

AIM To investigate the effect of paclitaxel-bivaludin interventional coating combination(LFN)on vascular restenosis and its molecular mechanism.METHODS In vivo animal experiment grouping:WT sham surgery group,WT vascular strain group,NRF2-/-vascular strain group,NF-κB-/-vascular strain group,WT vascular strain+LFN intervention group,NRF2-/-vascular strain+LFN intervention group,and NF-κB-/-vascular strain+LFN intervention group;In vitro cell experiment grouping:First batch of groups:Control group,LPS modeling group,LPS+LFN group,LPS+LFN+NF-κB knockdown group,LPS+LFN+IκB knockdown group,LPS+LFN+NF-κB over-expression group,and LPS+LFN+IκB over-expression group.Second batch of groups:Control group,LPS modeling group,LPS+LFN group,LPS+LFN+NRF2 knockdown group,LPS+LFN+Keap1 knockdown group,LPS+LFN+NRF2 over-expression group,and LPS+LFN+Keap1 over-expression group.The pathological changes of vascular tissue were detected using HE staining,the proliferation activity of vascular tissue was detected using immunofluorescence staining,the cell proliferation rate was detected using the CCK-8 method,the cell migration and invasion ability was detected using the Transwell method,and the gene and protein expressions of important targets and ligands of the NF-κB and NRF2/ARE pathways were determined in vascular tissues and cells using Q-PCR and Western blot.RESULTS In vascular strain model rats,LFN significantly inhibited intimal hyperplasia,which effectively prevented lumen restenosis and combated thrombosis simultaneously.LFN up-regulated the expressions of IκB,NRF2,NQO-1 and HO-1 genes and proteins compared with those in WT vascular strain group(P<0.05,P<0.01),but down-regulated the expressions of Keap-1 genes and proteins(P<0.05,P<0.01),which were reversed in NRF2-/-mutant rats.The antagonistic effect of LFN on the HCASMC model’s capacity for migration and invasion was affected by NF-κB,NRF2,with its ligands being knocked down or over-expressed.This also,to variable degrees,impaired LFN’s capacity to regulate the production of crucial proteins and genes in the NF-κB and NRF2/ARE pathways in cells.CONCLUSION LFN is efficacious in vivo against vascular restenosis and part of the effect is achieved by the unified regulation of the expressions of NF-κB and NRF2/ARE nuclear transcription factors and their essential ligands.